Chris:LabNotes/FateMapping/Calendar/2014/2014-10-30: Difference between revisions

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*Follow online published protocol (make sure to prep using specifications for smaller D5020 kit)
*Follow online published protocol (make sure to prep using specifications for smaller D5020 kit)
*Final elution = 10ul
*Final elution = 10ul
*'''Note: Can quantify bisulfite conversion recovery by using Qubit ssDNA assay'''

Revision as of 22:03, 5 November 2014

Bisulfite Conversion Test 3

Background

  • The previous two tests didn't seem to have worked (CircLigase didn't seem to have circularized either the sample or the positive control)
  • This time, I'll repeat the experiment but only use CircLigase II

Procedure

  • Below are the reactions I want to do for this experiment:
Rxn 1 (gDNA)    Bisulfite Conversion
Rxn 2 (gDNA)    Bisulfite Conversion -> T4 PNK -> CircLigase II -> Exo I/III
Rxn 3 (NTC)     Bisulfite Conversion
Rxn 4 (NTC)     Bisulfite Conversion -> T4 PNK -> CircLigase II -> Exo I/III

EZ DNA Methlyation-Direct

  • Use human gDNA (110ng/ul) for template for rxn's 1->4 (use 3.64ul to get 400ng of gDNA)
  • Follow online published protocol (make sure to prep using specifications for smaller D5020 kit)
  • Final elution = 10ul
  • Note: Can quantify bisulfite conversion recovery by using Qubit ssDNA assay